Enzymatic synthesis of oligosaccharide is generally performed through either of the following two methods: a method utilizing reverse reaction of enzymatic hydrolysis of oligosaccharide, or a method employing glycosyltransferase. From the viewpoints of synthesis yield, and applicability to synthesis of an oligosaccharide having a complicated structure, a synthesis method employing glycosyltransferase is considered more advantageous than a method utilizing reverse reaction of enzymatic hydrolysis of oligosaccharide. An increase in production of various glycosyltransferases through recent development of recombinant DNA technology contributes to realization of this synthesis technique.
However, sugar nucleotides (i.e., sugar donors), which are employed in such a glycosyltransferase-employing synthesis method, remain expensive (exclusive to some sugar nucleotides), and are available only in small amounts of reagent level. As has been reported, UDP-GalNAc is synthesized through, for example, a method employing combination of an enzymatic process and chemical acetylation, in which galactosamine is used as a starting material.
However, this method poses, for example, the following problems: (A) the reaction has been limited to only a small scale (laboratory scale); (B) difficulty is encountered in preparing an enzyme employed; (C) acetylation of galactosamine requires an ATP regeneration system, resulting in low overall reaction yield; and (D) UDP-galactosamine, when remaining in the reaction mixture, would cause a problem in terms of separation thereof from UDP-GalNAc, which is a target product. Therefore, this method has not necessarily been considered practical (Methods Enzymol., 28, 271-277, (1972), J. Org. Chem., 57, 152-157, (1992)).
Meanwhile, there has been reported a method in which UDP-GalNAc is synthesized from uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) by use of uridine 5′-diphospho-N-acetylglucosamine-4-epimerase (UDP-GlcNAc-4-epimerase) (WO 2002/050267). However, this method has the following problems: (A) only a small amount of UDP-GlcNAc-4-epimerase is present in animal tissues or microbial cells; and (B) since this enzymatic reaction employing UDP-GlcNAc-4-epimerase (which can be prepared through a recombinant DNA technique) is an equilibrium reaction, not only is conversion low, but also difficulty is encountered in separating UDP-GalNAc from UDP-GlcNAc (i.e., a substrate), which is not completely consumed and remains in the reaction mixture in a large amount. Therefore, this method remains problematic in practical use.
Patent Document 1: WO 2002/050267
Non-Patent Document 1: Methods Enzymol., 28, 271-277, (1972)
Non-Patent Document 2: J. Org. Chem., 57, 152-157, (1992)
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